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SAM 913.02

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 913

Supplemental Assay Method for the Detection of Coliforms in Antibody 

Products

Date: April 27, 2023

Number: SAM 913.02

Supersedes: SAM 911.01, April 14, 2017

Standard Requirement: 9 CFR 113.450(h)(2)(i)

Contact: Email: Methodsrequest.notification@usda.gov

 Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by 

USDA and does not imply its approval to the exclusion of other products that may be suitable.
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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Record and Report of Test Results

7. References 

8. Summary of Revisions

Appendix
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1. Introduction

This Supplemental Assay Method (SAM) describesthe test procedure used to detect coliform 

contamination in dried and liquid antibody products for oral administration, per title 9, Code of 

Federal Regulations (9 CFR), part 113.450(h)(2)(i). This test procedure uses Violet Red Bile

agar (VRBA) to detect coliform contamination.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Laminar-flow Class II biosafety cabinet (BSC)

2.1.2 Lab Armor® bead bath (set to 50°- 55°C)

2.1.3 Vortex mixer

2.1.4 30°- 35°C incubator

2.1.5 Analytical balance

2.1.6 Colony counter

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Escherichia coli (American Type Culture Collection (ATCC) #11775) or 

equivalent positive control culture

2.2.2 Violet Red Bile agar (VRBA) (Appendix), National Centers for Animal 

Health (NCAH) Media #10356 or as stated in the Outline of Production (OP)

from the biologics manufacturer.

2.2.3 Sterile water 

2.2.4 Glycerol diluent, 50%

2.2.5 Sterile pipettes, individually packaged

2.2.6 Sterile petri dishes, 100 x 15-mm
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2.2.7 Sterile culture tubes 

2.2.8 Lab coat or sterile sleeves and gloves

2.2.9 Spatulas

2.2.10 Weigh boats

2.2.11 Sterile wide mouth snap cap specimen containers

2.2.12 70% ethanol

2.2.13 4 x 4-inch sterile gauze pads

2.2.14 Pipetting aid

2.2.15 Micropipettors, 100-μL to 1.0-mL

2.2.16 Pipette tips, 100-μL to 1.0-mL

2.2.17 Sterile syringes with needles

2.2.18 Sarstedt vials, 2 mL

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge 

of aseptic biological laboratory techniques and preparation, proper handling and 

disposal of biological agents, reagents, tissue culture samples, and chemicals.

Personnel must also have knowledge of safe operating procedures and policies, as

well as training in the operation of the necessary laboratory equipment listed in 

Section 2.1.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate all equipment and instrumentation according to the 

manufacturer’s instructions and maintain according to standard operating 

procedures (SOPs).

3.2.2 Turn on the BSC at the beginning of the work week and leave on all week.
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3.2.3 Turn on the bead bath at least one day before use to allow the temperature 

to equilibrate. 

3.2.4 Monitor temperature of incubators, freezers, coolers, and bead baths 

according to SOPs.

3.3 Preparation of reagents/control procedures

3.3.1 Warm the samples and reference culture to 20°- 25°C (room temperature)

before rehydrating, if needed, to the volume listed on the vial or in the firm’s OP.

Thaw frozen products in the BSC immediately before testing.

3.3.2 E. coli reference stock culture is prepared according to the manufacturer’s 

instructions, suspended in 50% sterile glycerol, and mixed on a stir plate. Aliquots 

of 1.25 mL of prepared culture are dispensed into Sarstedt vials and stored at 

-75°± 5°C. For each test session, inoculate a petri dish with 10-100 CFU/0.1 mL 

to serve as a positive control.

3.3.3 Glycerol diluent, 50%: The 50% glycerol diluent is prepared by mixing 

equal parts of glycerol and 0.85% NaCl solution. Place 300 mL of diluent in a 

500-mL flask and sterilize at ≥121°C for 25 to 30 minutes or by following the 

manufacturer’s recommendations. Store at 20°- 25°C (room temperature) for 1 

year.

3.3.4 Negative/Technique Control: Inoculate one petri dish with 1 mL of the

water that was used as a diluent in the testing session. If no diluent is used in the 

testing session, use sterile water packaged in serum vials as the inoculum. Pour

15-20 mL of 50°- 55°C molten VRBA into each plate, swirl gently to mix and 

allow to solidify. Once the agar has hardened, invert the petri dish. Incubate this 

control plate with the serial test plates.

3.3.5 Preparation of the VRBA medium: On the day of the test, melt the VRBA 

in an autoclave for 5-10 minutes at 100°C. Place the bottles of melted media in 

the bead bath. Do not begin testing until the agar has cooled to 55°C. The 

temperature of the bead bath should be 50°- 55°C when the agar is ready. 

 3.4 Preparation of the sample

3.4.1 At least ten containers of final product are required for coliform purity 

 testing.

3.4.2 Samples are liquid or dried antibody products. When needed, sterile 

purified water in volumes specified on the product label or the diluent specified 

in the firm’s OP is used for rehydrating samples.
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3.4.3 Weigh samples of product on an analytical balance, if needed. Rehydrate 

samples of the product according to label or instructions from the firm's OP. 

Dissolve samples by stirring on a stir plate or shake with a vortex mixer. If 

sample does not readily go into solution after manipulating, allow the diluted 

sample to sit in a 30°- 35°C incubator for 1 hour (hr) ± 30 minutes (min). 

 

3.4.4 Label 10 plates for each serial with the sample number or name and the 

container number.

 4. Performance of the Test

4.1 Dress in a clean lab coat or sterile sleeves and gloves to perform purity testing.

4.2 Wipe down the interior surfaces of the BSC used for testing with 70% ethanol 

immediately prior to use and between testing each serial.

4.3 Place the necessary testing materials (micropipettors, pipet tips, 4 x 4-inch gauze 

squares, petri dishes, etc.) and the diluted (as stated in the firm’s OP) sample into the 

BSC.

4.4 Mix the sample thoroughly. Dispense 1 mL of inoculum from one sample 

container into a petri dish. 

4.5 Repeat Section 4.4 for the other nine sample containers for this serial.

4.6 Dispense 15-20 mL of 50°- 55°C molten VRBA medium into each of the 10

dishes for this serial. Swirl gently to mix and then allow the agar to cool in the BSC. 

Once the agar has hardened, invert the petri dishes and place the dishes in a 30°- 35°C

incubator for 24±4 hours (hrs). 

4.7 Repeat Sections 4.3 through 4.6 for the other serials of biologic to be tested.

4.8 Once all the test serials have been put on test, prepare the negative/technique 

controls for the testing session (see Section 3.3.4).

4.9 Once the purity portion of the test has been completed, prepare the positive 

control organism in an area that is separate and apart from the clean area where the purity 

test was conducted (see Section 3.3.2). 

4.9.1 Inoculate a petri dish with 10-100 CFU/0.1 mL of E. coli. 

4.9.2 Dispense 15-20 mL of 50°- 55°C molten VRBA medium into the plate. Swirl gently 

to mix and then allow the agar to cool in the BSC. Once the agar has hardened, 

invert the petri dish.
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4.10 All plates are incubated at 30°- 35°C for 24±4 hrs.

5. Interpretation of the Test Results

5.1 Criteria for a valid test:

5.1.1 There must be no growth in the negative/technique control and media

control plates.

5.1.2 The positive control plate is examined for coliform characteristic colony

growth and must contain a range of 10-100 CFU/0.1 mL. Characteristic growth is 

identified as dark red to purple colonies surrounded with or without a purple zone 

in the medium.

5.1.3 If these criteria are not met, the test is considered invalid or a no test (NT).

Products may be reported and released with a NT result if there is no reason to 

suspect an unsatisfactory (UNSAT) sterility result for that product.

5.2 After incubation, all plates are examined macroscopically for growth typical of 

coliforms. If coliform characteristic colony growth (see Section 5.1.2) appears on the 

positive control plate and does not appear on any negative control plates, the test is valid.

5.3 If no coliform colonies are detected on any of the sample agar plates, the serial is 

determined to be negative for coliform contamination and is satisfactory (SAT). If 

coliform characteristic colony growth is observed on any of the sample agar plates, the 

test is considered inconclusive (INC), and one retest (RT; see Section 5.5) to rule out 

faulty technique, may be conducted. If a RT is not conducted within 21 days, the serial is 

UNSAT by the first test.

5.4 If growth is observed on the negative or media control plate(s), or no 

characteristic growth is observed on the positive control plate, the test is a NT and may

be repeated without prejudice.

5.5 If a RT is ordered, double the number of samples used in the first test are 

required. If coliform characteristic colony growth is observed on any of the RT plates, the 

serial is UNSAT.

6. Record and Report of Test Results

Record and report results of the test(s) according to SOPs.
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7. References

Title 9, Code of Federal Regulations, part 113.450(h)(2)(i), U.S. Government Printing 

Office, Washington, DC. 

 Standard methods for the examination of dairy products. 1992, 16th Edition, Robert T. 

Marshall editor. American Public Health Association, Washington, DC. 

8. Summary of Revisions 

Version .02 

• The coversheet has been updated and document reformatted.
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Appendix

Violet Red Bile Agar (VRBA) – National Centers for Animal Health (NCAH)

 Media #10356

 Violet Red Bile Agar 41.5 g

QH20 1000.0 mL

Bring to a boil. Do not autoclave. Store at 2°- 5°C for up to a month.